High-affinity adaptors for switchable recognition of histidine-tagged proteins
High-affinity adaptors for switchable recognition of histidine-tagged proteins
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DOI：
10.1021/ja050690c
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发表时间：
2005-07-27
影响因子：
15
通讯作者：
Piehler, J
中科院分区：
文献类型：
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作者：
Lata, S;Reichel, A;Piehler, J
我们渴望创建化学识别单元，以高亲和力和稳定性结合寡聚组氨酸标签，作为选择性地将光谱探针和其他功能元件附着到重组蛋白上的工具。合成了几种含有 2-4 个次氮基三乙酸 (NTA) 部分的超分子实体，其中还含有一个氨基，荧光素与其偶联作为敏感的报告探针。这些多价螯合剂头 (MCH)（称为双、三和四-NTA）的特征在于它们与六组氨酸 (H6) 和十组氨酸 (H10) 标记靶标的相互作用。通过分析尺寸排阻色谱法观察到随着 NTA 部分数量的增加，结合稳定性显着增加。通过等温滴定量热法测定的结合焓几乎随着螯合剂头和标签之间可能的配位键的数量而增加。然而，为了获得完全相加的结合焓，寡聚组氨酸标签中需要大量过量的组氨酸。通过荧光去猝灭测量的 MCH/寡组氨酸复合物的解离动力学显示，与 mono-NTA 相比，稳定性提高了 4 个数量级，并且 tris-NTA 达到了亚纳摩尔亲和力。随着多价的增加，自由能的增加伴随着熵损失的增加，这归因于结合伙伴的高灵活性。可以预见这些 MCH 在将光谱探针和其他功能元件与重组蛋白进行非共价、高亲和力但可逆的束缚方面的多种应用。
We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.